Surface plasmon fluorescence microscopy characteristics and application to bioimaging

Furthermore, at the angle at which surface plasmon resonance occurs, it is shown that the emission of fluorescent beads collected through the metal layer results in an irregular point sp

SURFACE PLASMON FLUORESCENCE MICROSCOPY: CHARACTERISTICS AND APPLICATION TO BIOIMAGING

TANG WAI TENG

(B Eng (Hons), NUS)

A THESIS SUBMITTED FOR THE DEREE OF DOCTOR OF PHILOSOPHY

IN COMPUTATION AND SYSTEMS BIOLOGY (CSB)

SINGAPORE-MIT ALLIANCE

NATIONAL UNIVERSITY OF SINGAPORE

2010

Acknowledgments

First and foremost, thanks to my supervisors Prof Colin Sheppard and Prof Peter So for their guidance in my project, their patience and timely advice without which this work would not have been possible I am also grateful to Peter for the time I spent at

So Lab during my exchange at MIT And not forgetting many thanks to the members

of So Lab – Hyuk Sang Kwon, Euiheon Chung, Maxine Jonas, Jaewon Cha, Daekeun Kim, Heejin Choi, and Yanghyo Kim for making my stay a pleasant and memorable one Special thanks go to Euiheon who taught me about his standing wave TIRF system, and who has remained a wonderful collaborator and great friend Chapter six

is a result of our collaboration together with Yanghyo

I was fortunate to work with many wonderful people at Optical Bioimaging Lab – Kou Shan Shan, Naveen Balla, Shakil Rehman, Shalin Mehta, Elijah Yew, Zheng Wei, Si Ke, and Gong Wei Thanks to them for making the lab a fun and pleasant place to work in Our weekly Journal club was very enjoyable although our discussions always seemed to digress somehow Also, special thanks to Naveen and Sounderya Nagarajan for their help with cell culturing It was not always easy to find someone to coat the glass substrates I am thus indebted to Naganivetha Thiyagarajah and Xu Yingshun for their help with thin film deposition Finally, I am grateful to SMA for their support and funding over the past years for making this project possible Last but not least, I wish to thank my parents for their support while I complete my studies

Table of Contents

Table of Contents i

Summary iv

List of Tables vi

List of Figures vii

List of Symbols and Abbreviations x

CHAPTER 1: INTRODUCTION 1

1.1 Motivation 1

1.2 Literature Review 4

1.3 Background 6

1.3.1 Total Internal Reflection Fluorescence (TIRF) 6

1.3.2 Surface Plasmon Resonance (SPR) 12

1.4 An overview 17

CHAPTER 2: MODELING SURFACE PLASMON-COUPLED EMISSION MICROSCOPY 18

2.1 Surface Plasmon-Coupled Emission 18

2.2 Description of Angular Spectrum Representation 22

2.3 A Model for SPCEM 23

2.3.1 Excitation of dipole in the object space 23

2.3.2 Electric field in the image space 25

2.3.3 Addition of a linear polarizer in the detection path 32

2.3.4 Intensity in image space 33

CHAPTER 3: EXPERIMENTAL RESULTS OF SPCEM 35

3.1 Simulation Parameters 36

3.2 Radiation Characteristics of SPCEM 38

3.2.1 P-polarized emission and fluorescence quenching 38

3.2.2 Back-focal plane image of SPCEM 41

3.3 Point Spread Function Characteristics of SPCEM 42

3.3.1 Electric field distributions 42

3.3.2 Calculated intensity point spread function 45

3.3.3 Experimental set-up of SPCEM 45

3.4 Discussion 51

CHAPTER 4: OPTICAL TRANSFER FUNCTION OF SURFACE PLASMON-COUPLED EMISSION MICROSCOPY 53

4.1 Vectorial Optical Transfer Function 53

4.2 Comparison between TIRFM and SPCEM 57

4.3 Deconvolution for SPCEM 60

CHAPTER 5: OPTICAL COMPENSATION FOR SURFACE PLASMON-COUPLED EMISSION MICROSCOPY 71

5.1 Theoretical Basis 72

5.2 Modification to SPCEM 75

5.2.1 Spiral phase plate 76

5.2.2 Polarization mode converter 78

5.2.3 Comparison of SPP and PMC 80

5.3 Experimental Results 87

5.3.1 Modified SPCEM imaging 92

CHAPTER 6: STANDING-WAVE SURFACE PLASMON-COUPLED EMISSION MICROSCOPY 98

6.1 Background 98

6.2 Theory 99

6.2.1 Creating standing wave with p-polarized light 103

6.2.2 SW-SPRF algorithm and resolution 108

6.3 Experimental Results 110

6.3.1 SW-SPRF set-up 110

CHAPTER 7: CONFOCAL SURFACE PLASMON RESONANCE

FLUORESCENCE MICROSCOPY 117

7.1 SPR Excitation by Focused Beam 118

7.2 Theory for Confocal SPRF 125

7.3 Experimental Results 127

7.3.1 Generation of radially polarized beam 127

7.3.2 Confocal SPRF imaging 129

7.4 Detection with Conversion Element 132

CHAPTER 8: PERSPECTIVES AND CONCLUSION 139

BIBLIOGRAPHY 147

PUBLICATIONS 161

Summary

The characteristics of imaging through a metal-coated glass cover slip using the TIRF configuration are examined This configuration makes use of surface plasmons to excite fluorescent molecules as well as to collect the emission light Also known as surface plasmon coupled emission microscopy (SPCEM), the advantages of this method include better background rejection and reduced photobleaching due to decreased fluorescence lifetime Through back and front focal plane imaging of subdiffraction-limit fluorescent beads, the experimental characteristics of surface plasmon-coupled emission microscopy are elucidated Furthermore, at the angle at which surface plasmon resonance occurs, it is shown that the emission of fluorescent beads collected through the metal layer results in an irregular point spread function that has a donut-like morphology with multiple rings extending out Based on the vectorial Debye-Wolf method, a model for the microscope is derived and used to compare with the experimental results In addition, the vectorial OTF of the microscope is obtained and it is shown to have zero crossings and negative values which contribute to the donut shape morphology

Because of the distorted point spread function, point spread function engineering approaches or numerical deconvolution are necessary to compensate for the irregularity for practical use in cellular imaging Numerical deconvolution methods such as the Richardson-Lucy algorithm can compensate for the distortion However, an optical method is preferable because of less sensitivity to noise Due to

the anisotropic and highly p-polarized emission characteristics, we proposed to

engineer the point spread function using a conversion element, either a radial to linear mode converter or a spiral phase plate, which can compensate for the distortion Experimental results of compensated and uncompensated imaging using the conversion elements are presented It is shown that the conversion elements can restore the point spread function to one which is single-lobed, either by rotating the polarization of the emission light or by modifying its wavefront This change is beneficial for imaging Further improvements to the resolution of the microscope can

be achieved by using standing surface plasmon resonance waves A sub-diffraction limited resolution of 120 nm is demonstrated by illuminating the sample with a standing wave and synthesizing the final image from three images of different phases Finally, a confocal-based surface plasmon microscope is demonstrated which employs radially polarized illumination This results in a tightly focused spot and better excitation Together with a spiral phase plate in the detection path, its detection efficiency is shown to be improved without sacrificing the lateral resolution of the microscope

List of Tables

List of Figures

Figure 1-2 Total internal reflection with s- and p-polarized beams 9 Figure 1-3 Intensity at interface while varying the angle of incidence 10 Figure 1-4 Plot of the penetration depth of the evanescent field 11 Figure 1-5 A metal and dielectric half-space supporting surface plasmons 13

Figure 2-3 Excitation of dipole by p-polarized incident plane wave 24

Figure 2-5 Axis convention used in the derivation of the field in medium 3 26

Figure 3-5 Comparison of back-focal plane emission pattern of SPCEM 41 Figure 3-6 Electric field components for a perpendicular dipole 43

Figure 3-9 Experimental set-up of SPCEM 46 Figure 3-10 Comparison of theoretical and experimental point spread function 49

Figure 4-4 Flow chart for Richardson-Lucy deconvolution algorithm 63

Figure 5-6 A comparison of the cross-sectional profile of vectorial OTFs 83

Figure 5-12 Comparison of simulation and experimental results for SPP 93

Figure 5-13 Comparison of simulation and experimental results for PMC 94

Figure 6-1 Schematic diagram illustrating standing-wave excitation 102

Figure 6-7 Extended resolution imaging with standing-wave surface plasmons 115

Figure 7-3 Focused beam excitation with linearly polarized light 122 Figure 7-4 Focused beam excitation with radially polarized light 123

Figure 7-10 Comparison of power collected by pinhole for NA 1.45 objective 136

List of Symbols and Abbreviations

CCD Charged coupled device

FT2D[ ]• 2D Fourier transformation

OTF Optical transfer function

PMC Polarization mode converter

SPCE Surface plasmon coupled emission

SPCEM Surface plasmon coupled emission microscopy

TIRF Total internal reflection fluorescence

TIRFM Total internal reflection fluorescence microscopy

CHAPTER 1: I NTRODUCTION

1.1 Motivation

Presently, there exist many microscopy techniques available for visualizing tissues and biological samples, which can help scientific investigation and discovery in many areas such as in tissue pathogenesis as well as in probing of cellular processes Each

of these techniques varies in its ability to discriminate objects, for example, in terms

of lateral or axial resolution, depth of field, as well as their applicability or suitability for a particular imaging study The various imaging methods can be broadly classified into label-free techniques, and fluorescence techniques which make use of fluorescent probes introduced into the sample for imaging For label-free methods, there are phase contrast methods such as Differential Interference Contrast (DIC), methods based on interference such as Optical Coherence Tomography (OCT), or Second Harmonic Generation (SHG) microscopy which rely on the scattering or phase properties of the sample under investigation For fluorescence methods, techniques such as Confocal Laser Scanning Fluorescence Microscopy (CLSFM) and Multi-Photon Excitation (MPE) microscopy are commonly used for imaging due to advantages such as 3D

sectioning capability arising from the rejection of out-of-focus fluorescence or improved tissue penetration owing to longer wavelength used in MPE

For imaging biological specimens, an advantage of using fluorescent probes is that the signal-to-background ratio is relatively high since only proteins of interest are labeled Another advantage is the ability to study cellular processes that would otherwise be impossible with non-fluorescent techniques owing to the specificity achievable by tagging the proteins under study with fluorescence labels This makes fluorescence imaging one of the commonly used tools of biologists While CLSFM is commonly used in many biological studies, for situations where it is required to study processes near the cell membrane, a surface imaging technique called Total Internal Reflection Fluorescence Microscopy (TIRFM) (Axelrod 2001) is preferred due to its better axial resolution and improved background rejection This favorable characteristic of TIRFM stems from the fact that the fluorescent probes are excited by the evanescent waves when light at the glass and water interface is totally internally reflected due to the incident angle being larger than the critical angle of the medium Depending on the angle of incidence of the incoming beam, the depth of the evanescent waves is typically restricted to about 100 nm near the surface of the interface, and this contributes to the good axial resolution as compared to confocal microscopy which has an optical section of approximately 500 nm This quality of TIRFM makes it ideal for probing processes near the cell membrane For instance, this technique has been used to study actin dynamics (Sund and Axelrod 2000), exocytosis (Steyer et al 1997) as well as receptor-ligand binding (Schmid et al 1998)

At the same time as developments in fluorescence techniques rapidly progressed over the past few decades, interest in another area involving surface plasmons was renewed and gained momentum, especially over the past ten years after Ebbesen and coworkers’ discovery of the surface plasmons’ role in the extraordinary transmission

of light through sub-wavelength holes in metal (Ebbesen et al 1998) Since then, there has been a lot of research into using surface plasmons in negative index metamaterials and cloaking (Veselago 1968; Schurig et al 2006), in a perfect lens (Fang et al 2005; Pendry 2000; Smolyaninov et al 2007), and in optical waveguides (Bozhevolnyi et al 2001; Weeber et al 1999)

In addition, apart from the engineering applications listed above, studies into the application of surface plasmons in biology have also been ongoing For instance, the use of plasmonic gold nanoparticles has been investigated as a contrast agent in OCT due to the enhanced scattering caused by surface plasmons (Kah et al 2009; Lin et al 2005), and as a possible aid in photodynamic therapy due to their increased absorption cross-section (Huang et al 2007; Loo et al 2005) In the field of biosensing applications, developments utilizing surface plasmons have also been active For example, surface plasmon coupling between gold or silver particles has been demonstrated as an optical ruler, with the ability to monitor intramolecular distances

of approximately 70 nm without photobleaching unlike Förster Resonance Energy Transfer (FRET) (Sönnichsen et al 2005) More importantly, before these recent advances, surface plasmons have long been used for sensitive and quantitative biosensing applications such as for quantifying the kinetics of biomolecular interactions (Liedberg 1983; Fägerstam et al 1992) Likewise, the first surface

plasmon resonance based biosensor instrument was commercially available in 1992 and has been used in a number of key areas such as food analysis, drug discovery and biotherapeutics (Biacore 2010) Recent advances in the surface plasmon resonance (SPR) biosensor include improved sensitivity and resolution as well as higher throughput detection (Slavík and Homola 2007; Alleyne et al 2007; Piliarik et al 2005)

While progress has been made in many areas mentioned above, to date there have not been many studies devoted to understanding the application of surface plasmons to bioimaging, much less to fluorescence microscopy In many of the previous studies, the investigators mainly made use of the reflectivity and transmission characteristics of surface plasmons But the use of surface plasmons involving fluorescence excitation and emission in microscopy up till now has not been amply studied The aim of this thesis is therefore to study the effects of using surface plasmons specifically to fluorescence imaging and to elucidate the properties of such

an imaging modality in order to properly understand its characteristics and applicability for imaging purposes

1.2 Literature Review

In this section, we review some of the prior work that has so far been carried out to apply surface plasmons to microscopy and look at some of the applications that have been explored The use of surface plasmons in microscopy was initially proposed as a way to measure small variations on surfaces (Rothenhäusler and Knoll 1988; Yeatman and Ash 1987) This is due to the sensitivity of the surface plasmons to

small changes in the local dielectric environment of the surfaces In effect, small changes to the refractive index near a metal coated surface can give rise to a larger change in the reflectivity and transmission of light at that interface, therefore a high sensitivity can be achieved by simply detecting the change in the reflectivity of the light Experiments have shown the thickness sensitivity to be about 3Å while lateral resolution is in the order of 25µm Kano has proposed using a focused laser beam to excite surface plasmons and scanning the surface plasmon probe to form an image (Kano 2000; Kawata 2001) Somekh analysed and simulated two-photon and second harmonic surface plasmon microscopy with input beams of linear and circular polarizations (Somekh 2002) while Stabler proposed a Köhler illuminated configuration (Stabler et al 2004) Other input polarizations such as radial polarization have been recently demonstrated for single photon as well as two-photon imaging (Moh et al 2008, 2009) Recently, Giebel and coworkers made use of surface plasmons to quantify the distance of a cell membrane from the substrate (Giebel et al 1999) It is to be noted that the above works were based on the reflectivity and transmission characteristics of surface plasmon resonance, where one disadvantage is the long propagation length of surface plasmons which leads to a poor lateral resolution

Instead of using the reflectivity characteristics, surface plasmons can also be used to excite fluorescent molecules (Kitson et al 1996) For example, Yokota demonstrated single molecule fluorescence imaging of proteins using surface plasmon excitation (Yokota et al 1998) while Borejdo and coworkers used it with fluorescence correlation spectroscopy to study the dynamics of molecules (Borejdo, Calander, et al

2006) It has been shown that the enhanced electric field caused by surface plasmons

at the metal surface leads to a higher intensity of fluorescence signal (He et al 2006; Hung et al 2006) Furthermore, it was also shown that the metal surface contributes

to decreased lifetime of fluorescent molecules, which delays photobleaching (Chance

et al 1974; Drexhage 1970; Lukosz and Kuhn 1977; Vasilev et al 2004), and can be beneficial for fluorescence imaging Recently, surface plasmons were also used to image fluorescently labeled muscle fibers (Borejdo, Gryczynski, et al 2006; Burghardt et al 2006) The technique used was based on the TIRF configuration and the effect used is also commonly known as surface plasmon-coupled emission where the detection optics reside on the same side as the excitation optics (Gryczynski et al 2004b; Lakowicz et al 2003) So far, these experiments have been conducted using fluorescence in surface plasmon microscopy without having a clear comprehension of its imaging properties and characteristics It is therefore the aim of this thesis to study

in detail the imaging characteristics of surface plasmon microscopy

1.3 Background

In the following sections, the concepts of Total Internal Reflection Fluorescence (TIRF) and Surface Plasmon Resonance (SPR) are briefly reviewed

1.3.1 Total Internal Reflection Fluorescence (TIRF)

Total internal reflection can occur when light travels from a medium with a high refractive index to one with a low refractive index, for example from glass to air According to Snell’s Law which can be derived by considering the boundary

conditions for Maxwell’s Equations, the condition for this to happen is when the angle

of the incident light at the glass and air interface is greater than the critical angle (Hecht 2002) From Snell’s Law,

where n and i n denote the refractive index of the first (higher refractive index) and t

second (lower refractive index) medium, and θiand θt denote the angles the incident and refracted rays make with the normal at the interface of the media Therefore, when total internal reflection starts to occur, the refracted angle is at right angles to the normal and the critical angle is therefore defined as

where η is the ratio of the refractive indices of the media This is depicted in Figure 1-1 for two cases, one for which the incident angle is less than the critical angle, and the other where it is greater than the critical angle

When total internal reflection occurs, an evanescent field is set up at the interface between the two media The evanescent field is a localized and non-propagating field decaying in the axial direction but propagating in the transverse direction This can be seen in the following equations Assuming that the incident

light which has s-polarization is given by

From the above equations, it is evident that the evanescent field and the intensity

decay exponentially away from the interface with increasing z Hence the field is localized and does not propagate in the z direction; however it still propagates in the x

direction In addition, it is also possible for total internal reflection to occur when the

incident light is polarized (see Figure 1-2) The related expressions when using

p-polarized light are

i t

i p

For both s- and p-polarized incident light, the resulting evanescent field is

capable of exciting fluorophores which are placed at the interface (Axelrod 2001) It

n i

n t

θ i

z s-polarization

x

i k

s,t E

n i

n t

θ i

z p-polarization

x

i k

p,t E

Figure 1-2 Total internal reflection with s- and p-polarized incident beams result in

an evanescent field that decays exponentially with increasing z

between the x and z components, which implies having circular polarization in the x-z

plane The penetration depth of the evanescent field is given by the expression

0

4 n i sin i

λδ

a plot of the intensity at z = as a function of the angle of incidence for both s- and 0

Figure 1-3 Intensity at interface while varying the angle of incidence for s- polarized light (solid line) and p-polarized light (dash line)

p-polarized light with n i =1.5, n t =1.33 and λ0 =500nm while Figure 1-4 plots the penetration depth as a function of the angle of incidence These plots show that as the angle of incidence is increased, both the intensity and penetration depth decrease Hence, a compromise must be made between the desired intensity which affects the signal-to-noise and the penetration depth which affects the out-of-focus rejection While TIRF can be implemented using a prism, it is also commonly integrated into a microscope that uses an objective launched configuration owing to system simplicity and ease of use Many vendors such as Olympus and Leica have commercialized TIRF systems based on an objective launched configuration This configuration will be described in more detail in the next chapter

Figure 1-4 Plot of the penetration depth of the evanescent field as a function of

angle of incidence of s-polarized light.

1.3.2 Surface Plasmon Resonance (SPR)

Surface Plasmon Resonance (SPR) is a phenomenon in which free electrons near the surface of a metal-dielectric interface oscillate coherently in response to an applied electromagnetic field These charged oscillations are accompanied by a mixed transversal and longitudinal electromagnetic field which decays exponentially, and result in an enhanced field inside the dielectric medium which can be used to excite fluorescent probes As a variation to TIRF, the effect of SPR can also be employed to set up an evanescent field for exciting fluorescent probes near the interface layer This

is normally done by coating the cover slip with a thin layer of metal such as gold The advantage of SPR over TIRF is that the evanescent field set up with SPR is enhanced many times over that of TIRF and this contributes to more efficient excitation of the fluorophores for the same excitation power Furthermore, together with the wavelength dependency of SPR that tends to suppress background noise due to autofluorescence, these characteristics result in an improvement in the signal-to-noise ratio of SPR imaging

The criteria for supporting surface plasmons can be obtained by considering the transverse magnetic (TM) solution to a semi-infinite metal and dielectric half-space as shown in the illustration in Figure 1-5 It is also worthwhile to note that the transverse electric (TE) equations do not yield any solution, implying that surface plasmon

modes are only supported for a p-polarized incident beam and not an s-polarized

beam For z< , the fields are described by the following equations, 0

1 1

1

1

1 1

0 1 1

0 1

( )( )

ik x k z z

x

ik x k z x

2

2

2 2

0 2 2

0 2

( )( )

ik x k z z

x

ik x k z x

where k is the surface wave vector and x A A are field amplitudes 1, 2 k1z,k are the 2z

wave vectors normal to the interface and ε ε1, 2 are the permittivity of each medium respectively Considering boundary conditions at the interface z =0 results in the following conditions

,

z

z

A A k k

ε ε

ε ε ω

method, there exist two methods – the Otto configuration and the Raether configuration, of which the Kretschmann-Raether configuration is the most commonly used The solid line in the figure shows the effect of exciting surface plasmons using the ATR method In terms of configuration, the ATR coupler makes use of a prism geometry similar to prism-TIRF except with an additional metal layer coated on top of the glass prism as shown in Figure 1-7 Owing to the higher refractive index of the ATR coupler, the gradient of the light line is modified so that it

Kretschmann-is gently sloping and intersects with the SPR dKretschmann-ispersion curve, implying that surface plasmons can be excited Factors that affect the generation of SPR include the type of

metal layer used, the thickness of the metal layer, the angle of incidence of the

p-polarized incident beam and the refractive index of the dielectric medium (Raether 1988)

With the induced surface plasmons, one can employ them for exciting fluorescent probes placed at the metal-dielectric interface Alternatively, because of the sensitivity of the SPR effect to changes in the refractive index of the dielectric

Gold layer Surface plasmons

p-polarized beam

Prism

Figure 1-7 Geometry of the Krestchmann-Raether configuration for exciting surface plasmons

resulting in changes to the refractive index may be detected For example, by employing the Kretschmann-Raether configuration of Figure 1-7, the reflective curve

of SPR can be plotted for three layers of glass-silver-water for an excitation wavelength of 520 nm as shown in Figure 1-8 In the plot, when the refractive index

of the environment changes from 1.33 to 1.35, the reflectivity curve shifts to the right

by about 3 degrees Detection of this shift can allow for quantitative measurement of the refractive index change

Instruments based on this principle which makes use of SPR reflectivity shifts for measuring interaction between biomolecules are commercially available Furthermore, in the area of two dimensional imaging, the reflectivity characteristics of surface plasmons have been used in microscopy (Rothenhäusler and Knoll 1988; Yeatman and Ash 1987) Examples where application of surface plasmon microscopy Figure 1-8 A right shift in the SPR reflectivity curve due to a small change in the

refractive index of the environment from n=1.33 (solid line) to n=1.35 (dashed line)

(SPM) have been demonstrated include imaging of liquid crystals (Evans et al 1997), real-time imaging of birefringent samples (Tanaka and Kawata 2005) and imaging of cell-substrate contacts of living cells (Giebel et al 1999)

1.4 An overview

The rest of the thesis focuses on analyzing the characteristics of combining Surface Plasmon Resonance with the configuration of a TIRF microscope into a form of surface plasmon fluorescence microscopy also known as surface plasmon-coupled emission microscopy (SPCEM) for bioimaging Chapter two describes surface plasmon-coupled emission microscopy and derives the imaging theory as well as the point spread function In chapter three, experimental results are compared with simulation based on the derived theory The characteristics of the microscope are also reviewed In particular, the distorted point spread function characteristics are pointed out Chapter four analyzes the resolution of the microscope in terms of the optical transfer function Deconvolution methods that compensate for the distortion are analyzed Chapter five discusses optical methods to compensate for the aberrations to the point spread function Experimental results are also presented Chapter six introduces a technique to improve the resolution of the microscope based on standing-wave excitation In chapter seven, the scanning surface plasmon microscope is introduced and studied in detail Finally, chapter eight concludes the thesis

CHAPTER 2: M ODELING S URFACE

Both total internal reflection (TIR) and surface plasmon resonance (SPR) share similarities in terms of their set-up In addition, their evanescent fields can be used to excite fluorescent molecules In this chapter, the effect of combining SPR with the TIR set-up and detection of the emission light is discussed Furthermore, this is applied to microscopy using an objective-launched set-up and is called the SPCE microscope (SPCEM) The image formation and point spread function are then derived for the SPCE microscope

2.1 Surface Plasmon-Coupled Emission

The effect of surface plasmon-coupled emission (SPCE) was studied both theoretically and experimentally by a few investigators, where it was initially proposed as a high sensitivity detection measurement technique for fluorescence (Gryczynski et al 2004a; Lakowicz 2004; Lakowicz et al 2003) In SPCE, the set-up typically uses the Kretschmann-Raether configuration, which is similar to the TIRF

configuration Introduction of the metal layer on top of the prism allows excitation of the fluorescent molecules by surface plasmon resonance Such a combination allows more efficient energy transfer from photons to the fluorescent molecules than normal TIRF due to the coupling via surface plasmons The fluorescence emission is then detected on the same side as the excitation optics after the outcoupling of the emission light via surface plasmons SPCE has been used in many applications in biotechnology and biological measurements For example, the use of SPCE was demonstrated in DNA hybridization measurements (Malicka et al 2003), and in immunoassays (Matveeva, Z Gryczynski, I Gryczynski, and Lakowicz 2004; Matveeva, Z Gryczynski, I Gryczynski, Malicka, and Lakowicz 2004) where sensitive detection of myoglobin was demonstrated It was also used to extend fluorescence correlation spectroscopy to improve its signal-to-noise ratio (Borejdo,

Figure 2-1 Comparison of intensity enhancement at the interface between TIR (dashed line) and SPR (solid line)

Calander, et al 2006) One advantage of SPCE in exciting fluorescent molecules is that the evanescent field strength is typically stronger than normal TIRF at the surface plasmon resonance angle, where reflectivity is minimum and photons are efficiently coupled to the plasmons For example, Figure 2-1 compares the intensity enhancement at the metal interface for TIR and SPR excitation, assuming that gold is

used and that the incident beam is p-polarized The excitation wavelength is assumed

to be 532 nm, giving rise to a surface plasmon resonance angle of about 45 degrees

At this SPR angle, the enhancement is more than three times that of the TIR excitation with the same excitation angle This implies that for the same excitation power, SPCE gives rise to more efficient excitation of the fluorescent molecules than TIRF Other advantages of SPCE include small detection volume leading to less background signal, and reduction in photobleaching due to decreased fluorescence lifetime (Chance et al 1974; Drexhage 1970; Z Gryczynski et al 2006; Lukosz and Kuhn 1977; Vasilev et al 2004)

Apart from spectroscopic uses, SPCE can be used for 2D imaging by employing

an objective-launched microscope set-up as shown in Figure 2-2 In this configuration, the sample is deposited on a cover slip coated with a thin layer of metal such as gold which is commonly used as the metal layer due to its biocompatibility with cells and tissues This is then placed over a high numerical aperture (NA) TIRF objective lens Light from the excitation laser is focused at the back-focal plane of the objective and is emitted as a plane wave at an angle to the substrate By positioning the beam such that the incident angle is at the surface plasmon resonance angle, a strong evanescent plasmon field is set up to excite the fluorophores in the sample The

fluorescence emission is then outcoupled through via surface plasmons and is collected by the same high NA objective lens The image is finally captured by a CCD camera via an imaging lens This microscopy method is termed surface plasmon-coupled emission microscopy (SPCEM) and recently has been used to image muscle fibrils (Borejdo, Gryczynski, et al 2006; Burghardt et al 2006) An advantage of using SPCEM for surface imaging is the increased excitation efficiency due to the plasmon enhanced electric field at the dielectric interface Another advantage is the thinner detection volume, which is a result of the distance-dependent coupling of fluorophores to surface plasmons, and improved background rejection, both of which give rise to a better signal-to-noise ratio and higher image contrast (Z Gryczynski et

al 2006) In addition, another advantage is that the set-up allows simultaneous imaging of the fluorescence emission and measurement of the cell-substrate contacts

at the same time (Giebel et al 1999) This information can also be used to quantify

Metal coated

layer (gold) on

cover slip

Objective lens Emission

Excitation

Figure 2-2 An objective-launched set-up for SPCE imaging

ligand-receptor interactions However, SPCEM has not been fully understood in terms

of its emission out-coupling characteristics and one distinct disadvantage is that it makes multi-modal imaging with phase or DIC methods difficult More importantly, imaging characteristics of the set-up have not been fully described to-date In the subsequent sections, the full SPCEM imaging model and its point spread function is derived based on an angular spectrum representation This model provides a foundation for understanding the imaging characteristics of SPCEM and is important for quantitative or point spread function engineering applications

2.2 Description of Angular Spectrum Representation

Briefly, the angular spectrum of plane waves is a method based on an integral representation that is useful for studying the properties of wave fields In many wave-propagation problems, the analysis of diffraction at an aperture typically deals with wave fields in a half space and can be represented in the form (Born and Wolf 1999)

where this representation encompasses both homogenous and inhomogenous waves It

is noted that the above representations are in scalar terms The angular spectrum can

be generalized for the vector case and can be used to obtain an expression for the radiation of a dipole which is then used to derive a model for SPCEM

2.3 A Model for SPCEM

To understand the point spread function characteristics of SPCEM, a theoretical model can be developed by considering a fluorescent molecule placed on the gold-coated cover slip which is then imaged by the CCD camera The molecule is treated

as a point dipole that is excited by the incident light which then radiates at the emission wavelength; the angular spectrum of an emitting dipole can be determined The model is constructed in two steps First, the excitation of the dipole in the object

space by a p-polarized plane wave is considered Then, the dipole emission and the

subsequent propagation of the emission light through a 4f microscope system is modeled Finally, the intensity point spread function in the image space can be calculated These steps are shown in the following sections

2.3.1 Excitation of dipole in the object space

In SPCEM, dipoles on the metal-coated glass slide are excited by a p-polarized incident plane wave as shown in Figure 2-3 n 1 denotes the refractive index of the

object space, n is the refractive index of the metal layer and n is the refractive index

of the glass slide and immersion medium The dipole is placed at a distance d from the metal surface k 3,inc is the wave number of the incident plane wave in medium 3 intersecting the interface at an angle of θi from the surface normal, and is given as

3,inc 2 3/ inc

k = πn λ , where λ inc is the wavelength of the incident light in free space

As shown in the figure, the optical system used in SPCEM to excite the fluorophores by surface plasmons is similar to a TIRF microscope (D Axelrod 2003)

The excitation field E i of SPCEM is simply given by

where e x and e z denote the unit vectors in the x and z directions, and τ p,inc is the

three-layer Fresnel coefficient (Raether 1988) for p-polarized light given by

of the metal layer

Figure 2-3 Excitation of dipole by a p-polarized incident plane wave

2.3.2 Electric field in the image space

Figure 2-4 shows the typical set-up of a 4f system for SPCEM imaging The point dipole exp(p=µ
i tω ) at a distance d away from the first interface is excited by the

field calculated in Equation (2.4) and emits light at a different wavelength λ from the excitation wavelength λinc Region 2 is a thin metal layer of thickness t with an index

of refraction n 2 A high NA objective is placed in medium 3 and the tube lens in medium 4

Due to the highly polarized emission of SPCE and the high NA objective used,

a full vectorial formulation is derived for the model shown in Figure 2-4, since a scalar treatment of the model does not take into account these effects and is inadequate Using the vectorial Debye integral of Richards and Wolf (Richards and Wolf 1959; Wolf 1959), the field in the image space of the tube lens is given by

Figure 2-4 A schematic view of the SPCEM imaging process within a 4f optical system

To obtain the electric strength vector E in Equation (2.6), we first obtain the ′4

field emitted by the dipole in medium 1 which is subsequently transmitted into medium 3, and then propagated through the 4f system into medium 4 When considering SPCEM, it is important to note that the fluorophores are close to the

Figure 2-5 Axis convention used in the derivation of the field in medium 3

φ

metal surface, in the order of nanometers scale, which implies that the near-field emission cannot be ignored The procedure to obtain the field in medium 3 given below follows in part that given by Arnoldus and Foley (Arnoldus and Foley 2004) and takes into account the evanescent components It is noted that the field in medium

3 had also been derived by Hellen and Axelrod (Hellen and Axelrod 1987) in a similar way

Figure 2-5 shows the axis convention that is used for obtaining the expression for the dipole radiation in medium 3 The complex amplitude of the dipole source

field in an infinite medium with index of refraction n 1 is given by the equivalent vectorial form of the Weyl expansion for a dipole (Ford and Weber 1984)

1 2

ν ν

To obtain the field in medium 3, it is then necessary to decompose plane

waves which are propagating in the positive z direction into their p and s components,

so that it is possible to make use of the Fresnel transmission coefficient for the layer system First, the unit vectors for a cylindrical coordinate system whose radial